Nesher Technologies, Inc. (NTI) licensed from UCLA the IP for a revolutionary suite of ultrasensitive biodetection technologies involving alternating laser excitation (ALEX) fluorescence spectroscopy. These technologies were developed at the UCLA Single Molecule Biophysics Laboratory (headed by Prof. Shimon Weiss). NTI proposes to apply one of these technologies for very early-stage cancer diagnosis, monitoring of therapeutic response, assessment of outcome, prediction of recurrence, as well as basic cancer research. In the proposed implementation, dubbed alternating-laser excitation fluorescence-aided molecule sorting, or ALEX-FAMS, two recognition molecules are tagged with different color fluorescence dyes. Coincident detection of the two colors in a femto-liter confocal detection volume constitutes a positive target detection event. The proposal's broad long-term objective is development of highly multiplexed, mix-and-read, high- throughput solution phase assays for rapid and ultra-sensitive (femtomolar) quantification of protein and mRNA levels of tumor markers in serum, urine, and saliva (with a capacity of >10,000 markers per standard patient sample), overcoming drawbacks of current technologies and minimizing costs. Over the funding period, we will develop a protein-based assay using two high affinity fluorescently labeled antibodies to detect carcino-embryonic antigen (CEA) and demonstrate the quantitative nature of our methodology. Our specific aims are: [unreadable] 1. Expression and purification of a high-affinity antibody pair specific for CEA 2. [unreadable] 2. Conjugation of two different fluorescent dyes to these antibodies [unreadable] 3. Evaluation of CEA detection with ALEX-based single molecule fluorescence spectroscopy [unreadable] Development of ultra-sensitive, low-cost diagnostic assays that radically push the limits of current technologies by allowing simultaneous detection and accurate quantification of thousands of tumor markers from a single patient sample will aid both clinicians and cancer researchers in improving usefulness of tumor marker analysis. The prospect of monitoring the public on a routine basis for cancer-related changes in bodily fluids will significantly decrease mortality rates and and help fighting this deadly disease. [unreadable] [unreadable]